Pewarnaan Gram (Gram Staining-hsc
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Transcript of Pewarnaan Gram (Gram Staining-hsc
IBN PUTRA DWIJA, S.SI.,M.Biotech
Gram staining named after Danish Microbiologist Hans Cristian Gram (1853-1938)
1st used to differentiate of Pneumococci and Klebsiella pneumonia in lung specimen
Devised in 1882, but published in 1884 in Fortschitte der Medicin; 1884, Vol. 2, pages 185-189
Not the same with the method that we used today
Gram staining is differential staining which is differentiate microorganism (bacteria) into two large group (Gram positive and Gram negative) based on their chemical and physical properties of the cell walls
Gram staining is not used to staining Archaea because of variable response
Gram result can be used to decided empirical treatment, before culture of bacteria are finished as an definitive treatment
Gram staining can be used to decided of culture medium because Gram +ve/ -ve need different medium.
CELL WALL OF GRAM POS & NEG
CELL WALL IN GRAM +VE AND GRAM –VE BACTERIA Cell Wall Structures Gram
Positive organisms
Gram Negative organisms
Inner cytoplasmic membrane
Present Present
Peptidoglycan layer Thick Thin
Teichoic Acid Present Absent
Outer membrane layer Absent Present
Lipid A, LPS , Lipo-protien components
Absent Present
Peri-plasmic space Absent Present
Equipment for Gram stainingSlide glass (kaca slide/object glass)Wire loop (ose)Bunsen Gram setPencils glassMicroscope Immersion oilStaining rack
Gram setConsist of :
1. Primary stain (Crystal violet, methyl violet or Gentian violet)
2. Mordant (Gram's Iodine)/lugol 3. Decolourizer (ethyl alcohol, acetone or
1:1ethanol-acetone mixture) 4. Counterstain (Dilute carbol fuchsin, safranin
or neutral red)
The Gram Stain ProcedureStep 1 - Prepare a Smear
Watch what happens to the “Bacteria” at each step
“Bacteria”
Before take the specimen, make sure the loop is sterile
(burn wire loop on the Bunsen burner until red, cooling down a while) Remember : sterile your loop in every single step!!
Take one loop of bacterial culture/ specimen to be stained
Spread on the glass slide into proportional size
Allow to air dry. Heat fix by gently warming
The Gram Stain ProcedureStep 2 - Apply the Primary Stain
Flood the Smear with Crystal Violet
Allow to stand for 1-3 min
Rinse with water to remove excess stain
The Gram Stain ProcedureStep 3 - Apply the Mordant
Flood the Smear with Iodine / Lugol solution
Allow to stand 0,5-1 min
The Gram Stain ProcedureStep 4 - Rinse
Rinse with water to remove excess Iodine
The Gram Stain ProcedureStep 5 - Decolorize
Drip Decolorizer (80% Methanol +20% Acetone)/ alcohol 96% across the slide about 15-30 sec
The effluent should appear pale or clear
The Gram Stain ProcedureStep 6 - Rinse
Rinse with water to remove excess alcohol
The Gram Stain ProcedureStep 7 - Counterstain
Flood the slide with Safranin/Carbol Fuchsin solution
Let stand for 1-3 minutes
The Gram Stain
Step 8 - Rinse and Dry
Gram-Positive Gram-Negative
Rinse with water to remove excess stain
Dry with tissue, don’t swap the smear, let it air dry.
Observe your Gram slide under microscope with 100x lens
Add immersion oil during observation
Immersion oil make cell more clearly under microscope
Gram positive bacteria : blue-blackGram negative bacteria : red
If you want to make Gram stain from the specimen with a small amout of bacterial cell (e.q. urine, CSF etc) centrifuges its first to suspend the cell and make the smear as usually.
REMEMBER : Gram staining is only used for empiricallytreatment. Bacterial culture is the gold
standart
Thank you